7-alkyl- and 7-cycloalkyl-5-aryl-pyrrolo[2,3-d]pyrimidines - Potent inhibitors of the tyrosine kinase c-Src

Article abstract of DOI:10.1016/S0960-894X(01)00079-8

7-Substituted-5-aryl-pyrrolo[2,3-d]pyrimidines have been prepared starting from α-bromoacetophenones. These compounds represent a novel class of potent inhibitors of the tyrosine kinase pp60^c-Src with good specificity towards other tyrosine kinases (EGF-R, v-Abl).

Full text of DOI:10.1016/S0960-894X(01)00079-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 849–852
7-Alkyl- and 7-Cycloalkyl-5-aryl-pyrrolo[2,3-d]pyrimidines—
Potent Inhibitors of the Tyrosine Kinase c-Src
Leo Widler, Jonathan Green, Martin Missbach, Mira Susa and Eva Altmann*
Novartis Pharma Research, Arthritis and Bone Metabolism Therapeutic Area, CH-4002 Basel, Switzerland
Received 15 December 2000; revised 25 January 2001; accepted 2 February 2001
Abstract—7-Substituted-5-aryl-pyrrolo[2,3-d]pyrimidines have been prepared starting from a-bromoacetophenones. These com-
pounds represent a novel class of potent inhibitors of the tyrosine kinase pp60c-Src with good specificity towards other tyrosine
kinases (EGF-R, v-Abl). # 2001 Elsevier Science Ltd. All rights reserved.
A number of studies have provided compelling evidence
that the non-receptor protein tyrosine kinase pp60c-Src
(c-Src) plays a unique and essential role for osteoclast
function.1ꢀ4 Astransgenicmice lacking afunctional c-Src
gene develop normal numbers of osteoclasts, c-Src is
probably not involved in osteoclastogenesis. Rather, it
appears to be responsible for the phosphorylation of
cytoskeletal and/or docking proteins involved in trans-
location and exocytosis of vesicles during the resorption
process. Although c-Src is present in a broad variety of
cells, c-Src deficient mice do not show any overt patho-
logical signs other than osteopetrosis, a disease char-
acterized by lack of functionally active osteoclasts.1
Synthetic c-Src inhibitors reduce osteoclastic bone
resorption both in vitro5 and in vivo4 and should thus be
useful in the treatment of diseases such as osteoporosis
and tumor-induced osteolysis.
d]pyrimidine, IC50 for c-Src inhibition=0.1 mM), which
involves replacement of the N7-phenyl moiety by differ-
ent sugar surrogates (Fig. 1).6
Based on preliminary data, it was assumed that binding
of 1 occurs at the ATP binding site of the enzyme.
According to this model, the N7-phenyl ring is located
within the pocket that is usually occupied by the ribose
moiety of ATP. On the other hand the 5-phenyl ring
extends into a lipophilic pocket which is not exploited
by ATP, thus perhaps offering the opportunity to opti-
mize the selectivity profile of analogues of 1 by the
introduction of suitable ring substituents. Replacement
of the N7-phenyl substituent by open chain sugar ana-
logues as in 7 or substituted carbacycles as in 137 was
expected to improve potency and solubility of our lead
compound. The N7-substituent in compounds of type 7
can be viewed as an open chain sugar analogue, whereas
the substituted carbacycles in structures of type 13 repre-
sent more direct mimics of the ribose moiety of ATP.
We report herein on one of our optimization strategies
for our lead compound 1 (5,7-diphenyl-pyrrolo[2,3-
Figure 1.
*Corresponding author. Fax: +41-61-6966071; e-mail: eva.altmann@pharma.novartis.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00079-8

850
L. Widler et al. / Bioorg. Med. Chem. Lett. 11 (2001) 849–852
Optimization of the acyclic sugar moiety in analogues 7
was carried out by varying the distance between N7 and
the phenyl ring of the phenyl-alkanol moiety as well as
through the introduction of substituents at either or
both of the two phenyl rings. The synthesis of these
compounds is summarized in Scheme 1.
were obtained by a sequence of reactions similar to
those outlined in Scheme 1 for the synthesis of 7.
Treatment of a-bromoacetophenones 3 with sodium
azide gave the a-azidoacetophenones 8, which were
subsequently reduced to the corresponding amines.
Acetylation of the primary amino group provided sub-
stituted acetamides 9. Condensation of 9 with mal-
ononitrile furnished 2-amino-3-cyano-4-phenyl-pyrrole
10, which was then converted to the amidine 11 by
reaction with triethyl orthoformate followed by ammo-
nolysis of the resulting iminoester. Cyclization to the
desired 4-amino-5-aryl-7H-pyrrolo[2,3-d]pyrimidines 12
was achieved by treatment with sodium ethoxide.
Attachment of the (substituted) cycloalkyl moieties9 was
achieved via mesylate displacement (12!13a–k, 13m–p).
Reaction of amino alcohols 2 with a-bromoacetophe-
nones 3 produced the corresponding substituted amino
ketones 4, which were then converted to 2-amino-3-
cyanopyrroles 5 by treatment with malononitrile in the
presence of sodium ethoxide. Reaction of 5 with triethyl
orthoformate followed by ammonolysis resulted in
amidines 6, which could be transformed into the desired
pyrrolo-pyrimidines 7 by sodium ethoxide-induced
cyclization and subsequent acidic work-up.
Preparation of the cyclopentanol derivative 13l (Scheme 3)
involved reaction of 12 with the known epoxide 1510 to
give 16,10 followed by catalytic hydrogenation of the
olefinic double bond.
The synthesis of 7-cycloalkyl-pyrrolo-pyrimidines 13
(Scheme 2) in the first part involved the preparation of
4-amino-5-aryl-7H-pyrrolo[2,3-d]pyrimidines 12,8 which
Scheme 1. (a) DIEA, EtOH, 55 ꢁC, 25–39%; (b) CH2(CN)2, NaOEt, EtOH, 55 ꢁC, 89–97%; (c) (i) HC(OEt)3, 80 ꢁC; (ii) NH3/MeOH, rt, 25–54%;
(d) (i) NaOEt, EtOH, 80 ꢁC; (ii) HCl/MeOH, 26–80%.
Scheme 2. (a) NaN3, CH3N[(CH2)7CH3]3Cl (AliquatTM 366), toluene, 55 ꢁC, 41–87%; (b) H2, Pt/C, MeOH, HCl, 86–94%; (c) (Ac)2O, Et3N, THF,
rt, 81–89%; (d) CH2(CN)2, NaOEt, EtOH, 55 ꢁC, 61–94%; (e) (i) HC(OEt)3, 80 ꢁC; (ii) NH3/MeOH, rt, 68–82%; (f) NaOEt, EtOH, 80 ꢁC, 88–97%;
(g) R2-C4(5)H6(8)-OMes, K2CO3, 18-crown-6, DMF, 55 ꢁC, 24–96%.

L. Widler et al. / Bioorg. Med. Chem. Lett. 11 (2001) 849–852
851
Scheme 3. (a) (i) CH3COOOH, Na2CO3, CH2Cl2; (ii) Na2SO3, 11%; (b) (i) Pd[PPh3]4, DMSO/THF (1:1), rt; (ii) 12, 0 ꢁ!25 ꢁC (exclude light), 21%;
(c) H2, Pd/C (10%), MeOH, 98%.
All 7-substituted-5-aryl-pyrrolo[2,3-d]pyrimidines were
tested for c-Src inhibition in a liquid phase tyrosine
phosphorylation assay using chicken c-Src11 and the
synthetic substrate poly-Glu-Tyr (4:1).4 Inhibitors with
an IC50 below 0.5 mM were further tested for specificity
against EGF-R- and v-Abl-kinase and in a cellullar
assay measuring the c-Src mediated phosphorylation of
Fak in IC8.1 fibroblasts transfected with chicken c-Src
(Western blot analysis).4 The results are summarized in
Tables 1 (analogues 7) and 2(analogues 13).
of the 5-phenyl-pyrrolo[2,3-d]pyrimidine moiety generally
produces low nanomolar inhibitors of c-Src. Compounds
13c–e and 13k, i also exhibit potent cellular activity,
inhibiting cellular phosphorylation of Fak with sub-
micromolar IC50 values. Out of these latter analogues
13d possesses the most favorable overall profile, as it
inhibits the tyrosine kinase activity of the EGF receptor
and v-Abl ca. 100-fold less potently than that of c-Src.
In contrast, 13a–c, e, f, h, and i are significantly less
selective, in particular with respect to the inhibition of
v-Abl kinase. Attachment of a hydroxyl substituent to
the cyclopentane or cyclobutane moiety in the case of
an unsubstituted 5-phenyl ring (13l, m) slightly
improved activity against c-Src in the enzymatic assay
(3- and 5-fold, respectively) but resulted in a loss of cellular
potency for 13m. Compounds bearing substituents larger
than hydroxy on the cyclobutane ring were investigated in
combination with a meta-methoxyphenyl substituent at
the 5-position of the pyrrolo-pyrimidine ring system (13n–
p). Out of this series, compounds 13d and 13p emerged as
the most attractive analogues investigated, as they com-
bine potent cellular activity with a remarkable selectivity
profile versus EGF-R and v-Abl.12,13 In addition, 13p
possesses significantly improved aqueous solubility com-
pared to most other analogues investigated in this study.
As illustrated by the data summarized in Table 1, com-
pounds 7 with unsubstituted phenyl rings show sub-
micromolar c-Src activity only for n=0 (in the case of
the R-enantiomer) and n=1 (7b, c). Introduction of
substituents at the 4-position of the phenyl ring in the
phenyl-alkanol moiety of 7c (!7e), led to reduced inhi-
bitory potency. However, a dramatic increase in
potency could be achieved by introducing a 3-OH
substitutent on the 5-phenyl ring of 7e (!7g), which
resulted in an IC50 value for c-Src inhibiton of 42nM.
Most notably, 7g also exhibits a very favorable selec-
tivity profile and possesses submicromolar cellular
activity.12 Methylation of the 3-OH group on the 5-
phenyl ring as in compound 7h proved to be detrimental
for c-Src inhibition, resulting in a ca. 25-fold loss in
potency in comparison with 7g. Table 2summarizes the
c-Src inhibitory activity of analogues of structure 13. It
is immediately obvious that the presence of an unsub-
stituted cyclopentane or cyclobutane ring at the 7-position
In conclusion we have prepared a series of novel and
potent c-Src inhibitors. Some of these compounds exhibit
an excellent selectivity profile against EGF-R and v-Abl
tyrosine kinases.
Table 1. Inhibition of c-Src enzyme activity, cellular activity, and selectivity profile of inhibitors 7
Compd
R1
R2
n
c-Src (enzyme)a
c-Src (cell)b
EGF-Rc
v-Abld
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
7ae
7bf
7cg
7dg
7eg
7fg
7gg
7hg
H
H
H
H
H
H
3-OH
3-OMe
H
H
H
0
0
1
24
1
1
1
1
4.9
0.48
0.6
n.d.h
>5
n.d.h
n.d.h
0.44
n.d.h
14.4
13.22
0.34
9.7
n.d.h
n.d.h
10.5
n.d.h
h
H
n.d.
n.d.h
>10
OMe
OH
OMe
OMe
>1
1.9
0.0420.4
1
>5
n.d.h .2
3.4
n.d.h
0.68
aInhibition of c-Src enzyme activity in the liquid-phase tyrosine phosphorylation assay, c-Src concentration: 830 ng/mL, IC50 values are the mean of
2experiments carried out in duplicate, individual data points in each experiment were within a 3-fold range with each other.
bInhibition of c-Src mediated phosphorylation of Fak in IC8.1 fibroblasts.
cInhibition of epidermal growth factor receptor (EGF-R) tyrosine kinase enzyme activity.
dInhibition of v-Abl tyrosine kinase enzyme activity.
eS-Enantiomer.
fR-Enantiomer.
gRacemate.
hNot determined.

852
L. Widler et al. / Bioorg. Med. Chem. Lett. 11 (2001) 849–852
Table 2. Inhibition of c-Src enzyme activity, cellular activity, and selectivity profile of inhibitors 13
Compd
R1
R2
n
c-Src (enzyme)a
c-Src (cell)b
EGF-Rc
v-Abld
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
13a
13b
13c
13d
13e
H
H
H
H
H
H
H
H
H
20.14
20.08
20.02
20.002
20.001
1
1
1.8
1.2 0.57
0.2 0.11
0.46
0.2 0.08
0.50
0.14
4-OMe
4-OH
3-OMe
3-OH
H
4-OMe
4-OH
3-OMe
3-OH
H
6
0.2
0.3
0.17
5
0.17
0.01
13f
0.11
0.20
0.01
0.04
0.004
1
3
1.5
0.6
0.1
0.73
n.d.h
0.6
0.09
13g
13h
13i
13k
13le
13mf
13ng
13og
13pg
n.d.h
0.085
0.05
1
1
H
H
OH
0.45
0.3
1
20.05
1
1
1
1
0.005
0.2 0.79
0.04
H
OH
CH2OH
CONH(CH2)2OH
CH2NH(CH2)2OH
0.0204.3.892
0.03
0.024
2
3-OMe
3-OMe
3-OMe
20.4024
0.333
0.49
0.2
0.42
0.020.3
aInhibition of c-Src enzyme activity in the liquid phase tyrosine phosphorylation assay, c-Src concentration: 830 ng/mL, IC50 values are the mean of
2experiments carried out in duplicate, individual data points in each experiment were within a 3-fold range with each other.
bInhibition of c-Src mediated phosphorylation of Fak in IC8.1 fibroblasts.
cInhibition of epidermal growth factor receptor (EGF-R) tyrosine kinase enzyme activity.
dInhibition of v-Abl tyrosine kinase enzyme activity.
ecis-Racemate.
ftrans-Racemate.
gDiastereomeric mixtures.
hNot determined.
Acknowledgements
3134. (d) Wang, Y. D.; Miller, K.; Boschelli, D. H.; Ye, F.;
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dines have recently been reported as selective inhibitors of lck:
Arnold, L. D.; Calderwood, D. J.; Dixon, R. W.; Johnston,
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pyrimidine) has been reported: Taylor, E. C.; Hendess, R. W.
J. Am. Chem. Soc. 1965, 87, 1995. However poor reproduci-
bility of the described yields prompted us to look for an alter-
native route. The synthesis of 12 as shown in Scheme 1 was
adapted from: Johnson, R. W.; Mattson, R. J.; Sowell, J. W.
J. Heterocycl. Chem. 1977, 14, 383.
9. The synthesis of the 3-substituted cyclobutanols has been
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The authors thank Dr. H. Mett for providing us the
specificity assays and B. Fluckiger, D. Hurzeler, H.
Jeker, R. Klein, N.-H. Luong-Nguyen, M. Schaublin and
Y. Seltenmeyer for their excellent technical assistance.
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6. (a) Structurally different types of low molecular weight
inhibitors of c-Src have been reported, which were profiled as
anticancer agents: Dow, R. L.; Bechle, B. M.; Chou, T. T.;
Goddard, C.; Larson, E. R. Bioorg. Med. Chem. Lett. 1995, 5,
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12. IC50 values for inhibition of lck, a Src kinase family
member have been determined for selected compounds: 7g:
2.85 mM; 13d: 0.23 mM; 13p: 0.94 mM.
13. As pointed out by one of the referees, the apparent lack of
correlation between enzyme inhibition and cellular activity
might arise from inhibition of (unknown) additional or alter-
native cellular targets, which have not been addressed by our
work. Alternatively, the observed discrepancies may be caused
by differencies in cellular penetration for the individual com-
pounds. However, it should be noted that the IC50 determined
for the inhibition of autophosphorylation of EGF-R in a cel-
lular assay (Elisa) for a number of compounds (7g, 13c, 13e,
13i) were ꢂ8 mM, with one exception (13d, IC50=2.4 mM).